Jou nal o Medical Mic obiology
(2004), 53, 1–7 DOI 10.1099/jmm.0.45626-0
45626 &2004 SGM
P in ed in G ea B i ain
1
Co espondence
Ke in Ka anagh
[email p o ec ed]
Recei ed 5 Feb ua y 2004
Accep ed 29 Ap il 2004
Ampho e icin B enhances he syn hesis and elease
o he immunosupp essi e agen glio oxin om he
pulmona y pa hogen
Aspe gillus umiga us
Eme P. Ree es, Thomas Mu phy, Paul Daly and Ke in Ka anagh
Medical Mycology Uni , Na ional Ins i u e o Cellula Bio echnology, Depa men o Biology, Na ional
Uni e si y o I eland Maynoo h, Co. Kilda e, I eland
Exposu e o he pulmona y pa hogen Aspe gillus umiga us o ampho e icin B al e s memb ane
pe meabili y as indica ed by he escape o amino acids and p o ein om he mycelium. Ampho e icin
B exposu e o pe iods o 2–4 h also leads o inc eased elease o he immunosupp essi e agen
glio oxin in o he su ounding cul u e medium. Examina ion o he in acellula glio oxin concen a ion
ollowing exposu e o ampho e icin B indica ed ele a ed le els wi hin he hyphae as well as in he
cul u e medium – an e ec which was also e iden upon exposu e o A. umiga us o DMSO. These
esul s indica e ha in pa allel wi h he abili y o ampho e icin B o ac as a ungis a ic agen i can also
induce he syn hesis o glio oxin and acili a e i s elease by inc easing he pe meabili y o he ungal
cell memb ane. Inc eased syn hesis o glio oxin may esul om he commencemen o seconda y
me abolism in he p esence o ampho e icin B. The abili y o ampho e icin B o enhance he
syn hesis and elease o glio oxin may exace ba e he e ec s o he oxin and acili a e ungal in asion
o pulmona y issue.
INTRODUCTION
The ungus Aspe gillus umiga us is a pulmona y pa hogen
capable o inducing disease in hose wi h p e-exis ing
pulmona y mal unc ion (e.g. as hma, cys ic ib osis), disease
(e.g. ube culosis, lung cance ) o unde going immuno-
supp essi e he apy p io o o gan ansplan a ion
(Denning, 1996a; F ase , 1993; Daly & Ka anagh, 2001).
Th ee o ms o aspe gillosis a e ecognized clinically: sap o-
phy ic, alle gic and in asi e. In asi e aspe gillosis (IA) is he
mos se ious o m o disease as i in ol es he in asion o
iable issue and may p oduce a mo ali y a e o 80–95 %
(Denning, 1996b, 1998). IA has eme ged as an impo an
disease in ecen decades due o he use o agg essi e
immunosupp essi e he apy causing p olonged neu openia
in he ea men o cance and leukaemia (Daly & Ka anagh,
2001). Despi e agg essi e an i- ungal chemo he apy, dea h
due o IA usually esul s 7–14 days pos -diagnosis (Denning,
1996b).
A. umiga us p oduces a ange o seconda y me aboli es such
as glio oxin, hel olic acid and umagillin which may acili a e
i s g ow h and pe sis ence in he lung (Ami ani e al., 1995a;
Hogan e al., 1996). Ex ac s ob ained om spu um sols o
pa ien s wi h aspe gillosis damage human espi a o y epi he-
lial cells (Ami ani e al., 1995a). Subsequen analysis con-
i med ha glio oxin de i ed om A. umiga us was he oxic
agen and ha hel olic acid was also capable o comple e
cilios asis and epi helial cell dis up ion (Ami ani e al.,
1995b).
Glio oxin (C13H14N2O4S2, molecula mass 326.4) is an
epipoly hiodioxopipe azine (Wa ing & Bea e , 1996) which
displays immunosupp essi e p ope ies in i o (Su on e al.,
1994). Glio oxin is capable o inhibi ing mac ophage unc-
ion and adhe ence o plas ic su aces (Be ou e al., 2002;
Eichne e al., 1986) and may al e he immune esponse o
Aspe gillus as i can induce apop o ic cell dea h in mac o-
phages (Wa ing, 1990), cells o he spleen (B ai hwai e e al.,
1987) and he immune sys em (Su on e al., 1994). The
immunosupp essi e p ope ies o glio oxin ha e been e al-
ua ed o supp essing he whole body immune esponse
p io o o gan ansplan a ion (Su on e al., 1995). Glio oxin
is also capable o inducing cell dea h in a mu ine ib oblas ic
cell line (Pi a, 1994). Glio oxin has been de ec ed in samples
om animals (Richa d & DeBey, 1995; Richa d e al., 1996)
and humans (Shah e al., 1995) whe e i may acili a e ungal
pe sis ence and coloniza ion o issue. In addi ion, glio oxin
has been implica ed in he des uc ion o lung pa enchyma in
IA (Su on e al., 1996) and he pene a ion o blood essels in
angio-in asi e aspe gillosis (F ase , 1993)
Con en ional he apy o he con ol o aspe gillosis elies
upon he use o he polyene, ampho e icin B (Ellis, 2002) and
he azoles, i aconazole and luconazole (Canu o & Rode o,
2002). Ampho e icin B displays ungis a ic ac i i y and
unc ions by o ming ape u es in he cell memb ane, by
A icle numbe = 45626 Doc opic is: pa h i
Abb e ia ion: IA: In asi e aspe gillosis.
complexing he memb ane s e ol, e gos e ol (Abu-Salah,
1996). Each po e consis s o an annulus o eigh ampho-
e icin B molecules linked hyd ophobically o e gos e ol. The
hyd oxyl esidues o he polyene d ug ace inwa ds o gi e an
e ec i e po e diame e o 0.4–1.0 nm. Ampho e icin B–
e gos e ol po es allow he exi o in acellula cons i uen s
(e.g. ions and small molecula mass compounds) and he
en y o hyd ogen ions leading o inc eased cy oplasmic
acidi y (Cohen, 1998). While ampho e icin B can induce he
o ma ion o ape u es in he ungal cell memb ane, he e ec
may no be a al o he cell and ‘ eco e y’ may be possible
(Liao e al., 1999). I has been es ablished ha while a speci ic
concen a ion o ampho e icin B may inhibi eplica ion, as
indica ed by lack o ungal g ow h, speci ic cellula unc ions
(e.g. ans-memb ane po en ial, in acellula enzyme ac i i y
and memb ane in eg i y) s ill ope a e and, o e ime, he
ungus may esume he abili y o g ow and di ide (Liao e al.,
1999).
An al e na i e explana ion o he mode o ac ion o ampho-
e icin B has ecen ly been p oposed and sugges s ha , in
addi ion o he o ma ion o po es in he ungal cell
memb ane, ampho e icin B also al e s he pe meabili y o
he phospholipid bilaye by changing he memb ane phase,
i.e. inc easing he luidi y o he bilaye (Venegas e al. 2003).
Howe e , he dominan e ec o ampho e icin B appea s o
be he o ma ion o po es in he memb ane by complexing
e gos e ol.
The abili y o ampho e icin B o induce he elease o
in acellula cons i uen s in Candida albicans ia memb ane
ape u es has been es ablished p e iously (Ghosh & Ghosh,
1963) and aises he possibili y o he elease o low molecula
mass oxins om pa hogenic ungi. Consequen ly, he aim o
his s udy was o es ablish whe he exposu e o A. umiga us
o ampho e icin B could lead o he elease o a low molecula
mass oxin such as glio oxin; a p ocess which could ha e he
po en ial o exace ba e he immunosupp essi e and in asi e
abili ies o A. umiga us.
MATERIAL AND METHODS
A. umiga us
cul u e condi ions. A. umiga us ATCC 26933 (ob-
ained om he Ame ican Type Cul u e Collec ion) was used in his
s udy. Aspe gillus cul u es we e g own in minimal essen ial medium
Eagle (MEM) (Sigma Ald ich) o RPMI medium supplemen ed wi h
5 % ( / ) e al cal se um (Sigma Ald ich) a 37 8C and 200 .p.m., o up
o 4 days. S ocks we e main ained on mal ex ac aga (MEA) (Oxoid).
E ec o ampho e icin B on g ow h o
A. umiga us
.MEA pla es
con aining spo ula ing Aspe gillus colonies we e washed wi h 10 ml
0.1 % ( / ) Tween 80 (Me ck) in PBS (pH 7.2) (Sigma Ald ich) o
isola e conidia. Conidia we e washed wice in s e ile PBS, cen i uged
(1500 g, 5 min in a Beckman GS-6 cen i uge) and coun ed using an
haemocy ome e . Flasks con aining MEM (25 ml) we e inocula ed wi h
13105Aspe gillus conidia o gi e a densi y o 4 3103ml1and
incuba ed a 37 8C and 200 .p.m. Cul u es we e supplemen ed wi h
ampho e icin B a inal concen a ions o 0.04, 0.08, 0.16 and
0.32 ìgml
1. Flasks we e emo ed a each ime poin and he con en s
il e ed h ough a Wha man No. 1 il e in a Bu
¨chne unnel and ai -
d ied. A g ow h cu e was cons uc ed o d y ungal biomass e sus
incuba ion ime.
E alua ion o memb ane leakage. Se en y- wo-hou -old cul u es
(25 ml) o A. umiga us we e ha es ed by cen i uga ion (2056 g,20
min), washed h ee imes in PBS and he hyphal mass esuspended in
25 ml PBS. Ampho e icin B (0.04, 0.08, 0.16 o 0.32 ìgml
1) o DMSO
(Sigma Ald ich) (0.5 % / ) was added and he cul u es incuba ed a
37 8C and 200 .p.m. o a u he 1, 2 o 4 h. A each ime poin a cul u e
was emo ed and he con en s il e ed h ough a Wha man No. 1 il e .
The il a e was passed h ough a 0.45 ìM sy inge il e (Sa o ius) and
ee amino acids o p o ein we e measu ed as desc ibed below.
Amino acid concen a ion was de e mined by he ninhyd in colo i-
me ic me hod and is exp essed in e ms o aspa ic acid and glu amic
acid, which we e used as s anda ds. A ninhyd in (Sigma Ald ich)
solu ion (200 ìl; s ock: 0.35 g in 100 ml e hanol) was added o each
sample (1 ml) and hea ed o 95 8C o 4 min. A e cooling o oom
empe a u e in an ice ba h, he abso bance a 570 nm was eco ded on a
spec opho ome e (Beckman DU 640).
To de e mine he quan i y o p o ein eleased om he hyphal mass,
samples we e assayed using he B ad o d eagen (Bio-Rad), wi h BSA
(Sigma Ald ich) as s anda d.
Ex ac ion o glio oxin om
A. umiga us
cul u e il a e. Hyphae
o Aspe gillus we e emo ed om he MEM cul u e medium o RPMI
medium by il a ion and an equal olume (25 ml) o chlo o o m
(Hype Sol ; BDH) was added o he il a es. Following con inual
mixing o 30 min, he chlo o o m ac ion was collec ed and e apo-
a ed o d yness in a Bu
¨chi (B inkmann Ins umen s; Wes bu y, NY)
o o e apo a o . D ied ex ac s we e dissol ed in 250 ìl me hanol
(Hype Sol , BDH) and s o ed a 70 8C un il assayed.
Ex ac ion o glio oxin om hyphae o
A. umiga us
.The ex ac ion
o in acellula glio oxin om hyphae o A. umiga us was pe o med as
ollows: hyphae we e eco e ed om MEM cul u e medium (50 ml) o
RPMI medium (50 ml) by il a ion. Hyphae we e washed in PBS and
g ound o a ine powde unde liquid N2using a p e-chilled pes le and
mo a . The g ound hyphae we e esuspended in 10 ml 6 M HCl.
Chlo o o m (50 ml) was added and he mix u e s i ed a oom
empe a u e o 30 min. The sample was pou ed in o a sepa a ion
unnel and he glio oxin ex ac ed in he lowe chlo o o m laye .
Following chlo o o m e apo a ion, he d ied ex ac s we e dissol ed
in 250 ìl me hanol and le els o glio oxin quan i ied by Re e sed Phase-
HPLC.
Quan i ica ion o glio oxin by HPLC. Glio oxin was de ec ed by
Re e sed Phase-HPLC (Spec a-Physics). The mobile phase was 34.9%
( / ) ace oni ile (Hype Sol , BDH), 0.1 % ( / ) i luo oace ic acid
(Sigma Ald ich) and 65 % ( / ) deionized-dis illed wa e . Glio oxin
ex ac (20 ìl) was injec ed on o a C18 Hewle Packa d column. A
s anda d cu e o peak a ea e sus glio oxin concen a ion was
cons uc ed using glio oxin s anda ds (50, 100 and 200 ng ml1)
dissol ed in me hanol (Sigma Ald ich).
S a is ical analysis. All assays we e pe o med on h ee independen
occasions. Resul s p esen ed a e he mean s anda d e o . S a is ical
analysis we e pe o med using S uden ’s wo ailed - es wi h alues o
P,0.05 conside ed s a is ically signi ican .
E. P. Ree es and o he s
2
Jou nal o Medical Mic obiology
53
RESULTS
The e ec o ampho e icin B on he g ow h o
A.
umiga us
Cul u e medium was inocula ed wi h conidia o A. umiga us
a an ini ial densi y o 4 3103ml1and incuba ed as
desc ibed. An un ea ed cul u e and ou cul u es supple-
men ed wi h di e en concen a ions o ampho e icin B
we e used. The esul s (Fig. 1) indica e ha while he
concen a ions o ampho e icin B employed he e ac as
ungis a ic agen s o e he i s 24 h, he a e o g ow h o
he cul u es supplemen ed wi h 0.04 and 0.08 ìg ampho-
e icin B ml1is app oxima ely he same as he con ol o e
he 24–72 h pe iod. Following 96 h incuba ion, he ea ed
cul u es eached a mass o app oxima ely 65–70 % o ha o
he con ol. This expe imen demons a ed ha he concen-
a ions o ampho e icin B chosen o subsequen wo k we e
ungis a ic a he han ungicidal, as used clinically (Liao e
al., 1999).
To e i y ha he ampho e icin B concen a ions employed
in subsequen expe imen s we e ungis a ic a he han
ungicidal, 48 h cul u es o A. umiga us [hyphal mass
127.9 mg (d y weigh )] g own in MEM cul u e medium
we e supplemen ed wi h ampho e icin B a inal concen a-
ions o 0.16 o 0.32 ìgml
1and g own o a u he 24 h.
Hyphae we e ha es ed, washed wi h PBS and esuspended
in esh medium o 24 h in he absence o ampho e icin B.
The hyphal mass o he cul u e supplemen ed wi h 0.16 ìg
ampho e icin B ml1had inc eased o 155.4 mg (d y weigh )
while ha o he cul u e supplemen ed wi h ampho e icin B
a a concen a ion o 0.32 ìgml
1was 111.6 mg (d y
weigh ). This inding indica es ha ampho e icin B a
0.16 ìgml
1does no p e en subsequen g ow h o he
cul u e, and ha he 0.32 ìgml
1ampho e icin B ea men
does no cause wide-scale hyphal cell dea h, as he d y weigh
was s ill 87 % o he un ea ed con ol.
The e ec o ampho e icin B on elease o
in acellula cons i uen s om
A. umiga us
I has been es ablished p e iously ha ampho e icin B can
induce he elease o in acellula cons i uen s om pa ho-
genic ungi (Ghosh & Ghosh, 1963; Cohen, 1998). We sough
o es ablish whe he his occu ed in A. umiga us and o
de e mine whe he i con ibu ed o he enhanced elease o
low molecula mass compounds. Se en y- wo-hou -old
cul u es o A. umiga us we e exposed o ampho e icin B a
inal concen a ions o 0.04, 0.08, 0.16 o 0.32 ìgml
1o
0.5 % ( / ) DMSO o 1, 2 o 4 h and he elease o amino
acids and p o ein was moni o ed. DMSO was employed he e
as a posi i e con ol since i is known o al e he pe meabili y
o he cell memb ane (Yu & Quinn, 1998). The esul s show
ha exposu e o 0.16 o 0.32 ìg ampho e icin B ml1o
0.5 % DMSO o pe iods o 2 o 4 h lead o he elease o
ele a ed le els o amino acids compa ed o he ele an
con ols (Fig. 2). In he case o exposu e o A. umiga us o
0.32 ìg ampho e icin B ml1 o 4 h, 2.741 0.069 ìg
amino acid (mg hyphae)1was eleased.
Hyphal mass [mg (25 ml cul u e)–1]
Time (h)
0
5
10
15
20
25
30
35
40
45
024487296
Fig. 1. E ec o ampho e icin B on he g ow ho
A. umiga us
.Cul u e
medium was supplemen ed wi h ampho e icin B p io o inocula ion
wi h A. umiga us
conidia. d, Con ol; s,0
.4; j,0
.08; h,0
.16; n,
0.32 ìg ampho e icin B ml1. The hyphal mass was de e mined a
24 h in e als.
Ampho e icin B (µg ml–1)
Amino acids [∝
g (mg hyphae)–1]
0
0·5
1
1·5
2
2·5
3
Con
0·04
0·08
0·16
0·32
DMSO
Con
0·04
0·08
0·16
0·32
DMSO
Con
0·04
0·08
0·16
0.32
DMSO
1 h
2 h
4 h
**
*
*
**
*
*
*
Fig. 2. E ec o ampho e icin B on elease o amino acids om
hyphae o
A. umiga us
. Cul u es o
A. umiga us
we e supplemen ed
wi h ampho e icin B and he elease o amino acids was assessed
using he ninhyd in assay. DMSO was used as a posi i e con ol a a
concen a ion o 0.5 % ( / ). *, S a is ically signi ican (
P
,0.05)
di e ence compa ed o he ele an con ol.
Ampho e icin-B-media ed elease o glio oxin om
A. umiga us
h p://jmm.sgmjou nals.o g 3
Enhanced p o ein elease om A. umiga us was also e iden
a e exposu e o ampho e icin B o 2 o 4 h (Fig. 3). In his
case he g ea es elease occu ed when 0.32 ìg ampho e icin
Bml
1was employed o 4 h (35.57 2.56).
Release o glio oxin om
A. umiga us
exposed o
ampho e icin B
Ampho e icin B can induce he elease o amino acids and
p o ein om cul u es o A. umiga us o e a ela i ely sho
ime pe iod (Figs 2 and 3). Since glio oxin is a low molecula
mass dipep ide i could escape om A. umiga us in he same
manne as amino acids and p o eins. Cul u es o A. umiga us
which had been g own o a pe iod o 72 h o a hyphal mass o
29 mg we e supplemen ed wi h ampho e icin B a inal
concen a ions o 0.04, 0.08, 0.16 o 0.32 ìgml
1and he
amoun o glio oxin in he cul u e il a es was assessed a
a ious ime poin s a e he addi ion. A e 1 h, he con ol
cul u e demons a ed a glio oxin concen a ion o
210.336.1 ng (mg hyphae)1(Fig. 4). Those cul u es
supplemen ed wi h 0.16 o 0.32 ìg ampho e icin B ml1
showed ele a ed le els o glio oxin in he cul u e supe -
na an s, pa icula ly a e 2 and 4 h. The highes concen a-
ion o glio oxin [506.624.7 ng (mg hyphae)1] was
de ec ed in he cul u e supplemen ed wi h 0.32 ìg ampho-
e icin B ml1a e 2 h incuba ion (Fig. 4).
E ec o DMSO on glio oxin elease by
A. umiga us
Ampho e icin B unc ions by binding e gos e ol in he ungal
cell memb ane and c ea ing po es h ough which in acellu-
la cons i uen s may escape (Abu-Salah, 1996). I was
pos ula ed ha he ele a ed le els o glio oxin obse ed in
ampho e icin-B-supplemen ed cul u es could be due o he
inc eased pe meabili y o he ungal cell memb ane. To es
his hypo hesis 72-h-old cul u es o A. umiga us we e
supplemen ed wi h DMSO, which also has he abili y o al e
he pe meabili y o cell memb anes, bu in a di e en manne
o ampho e icin B (Yu & Quinn, 1998). The esul s demon-
s a ed a signi ican inc ease in glio oxin in he cul u e
medium a e 2 h in hose cul u es supplemen ed wi h
0.5 % DMSO (Fig. 5). Following 4 h exposu e o DMSO,
he glio oxin concen a ion in he cul u e medium supple-
men ed wi h 0.5 % DMSO eached a alue o
431.14 23.1 ng (mg hyphae)1.
Examina ion o in acellula and ex acellula
concen a ion o glio oxin ollowing exposu e o
A. umiga us
o ampho e icin B o DMSO
The ele a ed le els o glio oxin in ampho e icin-B- o
DMSO-supplemen ed cul u es may be due o inc eased
memb ane pe meabili y. Al e na i ely, he wo agen s could
exe a ungis a ic e ec and his migh , in u n, lead o he
inc eased syn hesis o seconda y me aboli es such as glio ox-
in. To asce ain whe he his was he case he glio oxin
concen a ion in supplemen ed medium and in he hyphae
o A. umiga us was de e mined. In his case cul u es we e
g own o 48 h and hen supplemen ed wi h he ele an
concen a ion o ampho e icin B o DMSO o a u he
24 h. The esul s e ealed enhanced le els o glio oxin in he
hyphae as well as in he cul u e medium o hose cul u es
supplemen ed wi h ampho e icin B o DMSO (Fig. 6).
The in a-hyphal glio oxin concen a ion in he cul u e
Ampho e icin B (µg ml–1)
P o ein [∝
g (mg hyphae)–1]
Con
0·04
0·08
0·16
0·32
DMSO
Con
0·04
0·08
0·16
0·32
DMSO
Con
0·04
0·08
0·16
0·32
DMSO
0
5
10
15
20
25
30
35
40
45
1 h
2 h
4 h
*
*
**
*
*
*
**
*
*
*
*
*
*
Fig. 3. P o ein elease om
A. umiga us
ollowing exposu e o
ampho e icin B. Cul u es we e supplemen ed wi h ampho e icin B
and he elease o p o ein om hyphae was measu ed. DMSO was
used as a posi i e con ol a a concen a ion o 0.5 % ( / ). *,
S a is ically signi ican (
P
,0.05) di e ence compa ed o he ele-
an con ol.
Ampho e icin B (µg ml–1)
Glio oxin [ng (mg hyphae)
–1
]
1 h
2 h
4 h
0
100
200
300
400
500
600
Con
0·04
0·08
0·16
0·32
Con
0·04
0·08
0·16
0·32
Con
0·04
0·08
0·16
0·32
*
*
*
*
***
Fig. 4. Release o glio oxin om
A. umiga us
cul u es supplemen ed
wi h ampho e icin B. The concen a ion o glio oxin in ampho e icin-B-
supplemen ed cul u es was assessed by HPLC analysis. *, S a is ically
signi ican (
P
,0.05) di e ence compa ed o he ele an con ol.
E. P. Ree es and o he s
4
Jou nal o Medical Mic obiology
53
supplemen ed wi h 0.16 ìg ampho e icin B ml1was 732.1
37.9ngmg
1compa ed o he con ol which showed a
glio oxin concen a ion o 88.64.9ngmg
1. Simila ly, he
glio oxin concen a ion in he MEM cul u e medium was
273.511.9ngml
1compa ed o 146.14.5ngml
1in
he con ol. In he case o he cul u es supplemen ed wi h
0.5 % ( / ) DMSO, he glio oxin concen a ion in he cul u e
medium was 227 12.1ngml
1(con ol: 146.114.5
ng ml1) while ha in he hyphae was ound o be
405.520.2ngmg
1compa ed o 88.64.9ngmg
1in
he ele an con ol. In he case o A. umiga us g own in
RPMI medium (Fig. 6) he ex acellula glio oxin concen a-
ion was 39.47 1.2ngml
1in he con ol whe eas i was
205.19 8.2ngml
1when he cul u e was supplemen ed
wi h ampho e icin B a a inal concen a ion o 0.16 ìgml
1.
The in acellula glio oxin concen a ion inc eased om
3.63 0.13 ng (mg hyphae)1in he con ol o 41.4
0.74 ng (mg hyphae)1in he ampho e icin-B- ea ed cul-
u e. Al hough di e en le els o glio oxin we e p oduced by
A. umiga us in MEM and RPMI cul u e media, he abili y o
ampho e icin B o inc ease he in acellula and ex acellula
glio oxin le els was obse ed in cul u es g own in bo h
media.
DISCUSSION
In ec ion by A. umiga us is a se ious h ea o he heal h o
pa ien s immunocomp omised as a esul o disease, pul-
mona y mal unc ion o medical he apy (Daly & Ka anagh,
2001) and, in he case o IA, may ha e a mo ali y a e o 85–
90 % (Denning, 1996b). Pa o he abili y o A. umiga us o
colonize he lung is i s capaci y o supp ess he local immune
esponse (Hogan e al., 1996). This can be achie ed h ough
he ac ion o oxins and enzymes, and he abili y o glio oxin
o acili a e coloniza ion is well cha ac e ized (Su on e al.,
1995 & 1996). Glio oxin may supp ess he unc ion o
pulmona y mac ophages and also a ec he gene al immune
esponse o pa hogens by des oying cells o he spleen and
he immune sys em (Su on e al., 1994).
One o he p incipal d ugs used o comba aspe gillosis is he
polyene ampho e icin B, which c ea es po es in he ungal
cell memb ane h ough which in acellula cons i uen s
escape (Abu-Salah, 1996; Cohen, 1998). I was pos ula ed
ha such po es migh also se e o allow glio oxin o escape
om he hyphae o A. umiga us, which could exace ba e
glio oxin-media ed damage o pulmona y issue and he
supp ession o he immune esponse in he in ec ed pa ien .
As a consequence we sough o de e mine he esponse o
hyphae o A. umiga us o ampho e icin B in e ms o he
syn hesis and elease o glio oxin.
Using ungis a ic concen a ions o ampho e icin B (Fig. 1) i
was es ablished ha exposu e o cul u es o A. umiga us o
ampho e icin B inc eased he elease o amino acids (Fig. 2)
and p o ein (Fig. 3) 2–4 h a e addi ion. These inding
sugges ed ha ampho e icin B was inducing he elease o
in acellula cons i uen s om he ungal hyphae, possibly
h ough he c ea ion o po es in he cell memb ane. Ele a ed
1 h
4 h
2 h
Glio oxin [ng (mg hyphae)
–1
]
0
50
100
150
200
250
300
350
400
450
500
DMSO (%)
Con
0·25
0·5
1·0
Con
0·25
0·5
1·0
Con
0·25
0·5
1·0
*
*
*
*
*
*
*
Fig. 5. E ec o DMSO on elease o glio oxin om
A. umiga us
.
Cul u es o
A. umiga us
we e supplemen ed wi h DMSO and he
elease o glio oxin o e a 4 h pe iod was assessed by HPLC analysis.
*, S a is ically signi ican (
P
,0.05) di e ence compa ed o he
ele an con ol.
0
50
100
150
200
250
300
MEM
AmpB
(0·08µg ml–1)
MEM
Con ol
MEM
AmpB
(0·16µg ml–1)
MEM
DMSO
(0·5%)
0
100
200
300
400
500
600
700
800
900
RPMI
AmpB
(0·16µg ml–1)
RPMI
Con ol
*
*
*
*
+
+
+
+
Ex acellula glio oxin [ng (ml cul u e)–1]
In acellula glio oxin [ng (mg hyphae)–1]
Fig. 6. In a- and ex acellula concen a ion o glio oxin in cul u es
supplemen ed wi h ampho e icin B o DMSO. Cul u es o
A. umiga us
we e supplemen ed wi h ampho e icin B (0.08 o 0.16 ìgml
1)o
DMSO (0.5 % / ). Ex acellula (open ba s) and in acellula ( illed
ba s) glio oxin was ex ac ed and quan i ied by HPLC. S a is ically
signi ican di e ences (
P
,0.05) compa ed o he ele an con ols
a e indica ed by * o +.
Ampho e icin-B-media ed elease o glio oxin om
A. umiga us
h p://jmm.sgmjou nals.o g 5

le els o glio oxin we e also de ec ed in media o cul u es
supplemen ed wi h ampho e icin B (Fig. 4) o DMSO (Fig.
5). DMSO is commonly used as a sol en in biological science
and can al e cell pe meabili y by a ec ing he s uc u e and
in e ac ion o lipids in memb anes (Yu & Quinn, 1998;
Go deliy e al., 1998) which may explain he elease o amino
acids, p o ein and glio oxin obse ed he e.
Gi en he p e iously es ablished abili y o ampho e icin B o
induce he elease o in acellula cons i uen s om C.
albicans (Ghosh & Ghosh, 1963), i appea ed ha his
mechanism migh also be ope a ing he e o acili a e he
elease o glio oxin. Howe e , when he in acellula con-
cen a ion o glio oxin was asce ained i was ound o be
ele a ed, along wi h aised le els de ec ed in he cul u e
supe na an . In he case o MEM cul u es supplemen ed wi h
0.08 o 0.16 ìg ampho e icin B ml1, he in acellula
concen a ion o glio oxin ose om 88.64.9ngmg
1 o
217.38 18.8ngmg
1and 732.137.9ngmg
1, espec-
i ely (Fig. 6). This e ec was also appa en in RPMI-g own
cul u es o A. umiga us.
The inc eased concen a ion o glio oxin in cul u e supe -
na an may be explained by he well es ablished abili y o
ampho e icin B o inc ease he pe meabili y o he ungal cell
memb ane ia he c ea ion o po es (Ghosh & Ghosh, 1963;
Cohen, 1998). Howe e , he ele a ed le el o in acellula
glio oxin could also con ibu e o he highe le els de ec ed
in he cul u e. The abili y o ampho e icin B o inc ease he
syn hesis o glio oxin may be media ed h ough i s ungi-
s a ic abili ies. When applied o cul u es o C. albicans,
ampho e icin B hal s eplica ion bu he cells emain iable
(Liao e al., 1999) and he ungus may eco e he abili y o
di ide in he absence o he polyene. Hal ing he cell’s g ow h
may o ce he p oduc ion o seconda y me aboli es such as
glio oxin. In e ec ampho e icin B may ha e wo dis inc
e ec s on he ungal cell: (i) The ungis a ic e ec may hal
eplica ion, lea ing he cell ali e bu capable o syn hesizing
seconda y me aboli es such as glio oxin; (ii) The abili y o he
polyene o c ea e ape u es in he ungal cell memb ane
allows he escape o small molecula mass compounds,
including glio oxin. The consequence o ampho e icin B
exposu e a he cellula le el would be ele a ed syn hesis and
elease, ia memb ane ape u es, o glio oxin.
Al hough DMSO has a di e en p ima y e ec on cell
memb anes (Yu & Quinn, 1998), i also induces ele a ed
syn hesis and elease o glio oxin. In he case o exposu e o A.
umiga us o DMSO, he ele a ed le els o glio oxin in he
supe na an may be due o he inc eased pe meabili y o he
ungal cell memb ane. The leakage o me aboli es may hinde
he cell’s abili y o di ide, empo a ily allowing i syn hesize
seconda y me aboli es such as glio oxin.
The wo k p esen ed he e indica es ha when exposed o
ampho e icin B in i o A. umiga us p oduces and sec e es
he immunosupp essi e agen glio oxin a an ele a ed a e.
Ampho e icin B he apy has many undesi able side e ec s
(Cohen, 1998) and new o mula ions a e designed o
minimize he oxici y owa ds hos issue (Ha sel & Bola d,
1996). Apa om i s di ec e ec on he hos , ampho e icin
B may also induce he syn hesis and elease o seconda y
me aboli es by A. umiga us which may con ibu e o g ea e
pulmona y damage and dec eased immune unc ion bo h
locally and sys emically. While he wo k p esen ed he e
examined he esponse o A. umiga us o ampho e icin B
in an in i o model, in i o e alua ion o his phenomenon
may be equi ed o ully assess i s clinical signi icance. Recen
wo k has indica ed ha ungi can su i e exposu e o
ela i ely high le els o ampho e icin B, main ain ce ain
cellula unc ions and po en ially esume eplica ion (Liao e
al., 1999). This s udy has es ablished ha ampho e icin B
may induce he syn hesis and elease o glio oxin om A.
umiga us. This phenomenon has he po en ial o con ibu e
o ele a ed le els o pulmona y damage (Eichne e al., 1986;
Su on e al., 1996) and immunosupp ession (Su on e al.,
1994, 1995) associa ed wi h his oxin.
ACKNOWLEDGEMENTS
This wo k was conduc ed independen ly o any pha maceu ical
company wi h in e es s in an i- ungal d ug de elopmen o ma ke ing.
This wo k was suppo ed by unding om he Highe Educa ion
Au ho i y h ough PTRLI 3.
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Ampho e icin-B-media ed elease o glio oxin om
A. umiga us
h p://jmm.sgmjou nals.o g 7
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